Immunotherapeutic combination for the treatment of tumors that over-express receptors with tyrosine kinase activity

ABSTRACT

The present invention is related to the field of immunology and more specifically to cancer immunotherapy, particularly with immunotherapeutic combinations and treatment methods to prevent tumor cell growth and/or to eliminate those cells.  
     The methods described in the present invention are based on the blockade of receptors with protein kinase activity in tyrosine residues (Receptor Tyrosine Kinases, RTK) and of ligands for those receptors.  
     Immunotherapeutic combinations are described that cause the blockade of RTK receptors and/or their ligands, by means of a combination of passive and active immunotherapy. The referenced procedures can be applied to patients in different clinical stages with tumors of epithelial origin that over-express those receptors. The combination of active and passive immunotherapy can be simultaneous or sequential independent of whether the therapeutic procedure will be used in patients with advanced disease or as adjuvant therapy.

[0001] The system of the EGF receptor (EGF-R) and its ligandsconstitutes a molecular complex whose interaction regulates in aspecific way cellular growth and its impact has been demonstrated in theuncontrolled growth of tumors of epithelial cell origin. Duringtumorigenesis the paracrine and autocrine control of EGF-R activation isderegulated, due to growth factor over-production, because of the highrate of synthesis and/or receptor mutations.

[0002] The EGF-R is a transmembrane glycoprotein with 1186 amino acidsand 170 kD molecular weight that it is broadly expressed in normaltissues. It has been implicated in several stages of embryogenicdevelopment.

[0003] The binding of its specific ligands, EGF or TGF-alpha, inducesreceptor dimerization, as well as heterodimerization with other membersof the ErbB family, like HER-2 (Cohen B D et al. (1996) J Biol Chem271:7620-7629). The binding of ligand to receptors releases a cascade ofintracellular signals (Ullrich T O and Schlessinger J (1990) Cell61:203-212) that drives cellular growth and differentiation.Overexpression of the receptor occurs in some types of cancers, mainlyof epithelial origin, which has been a target for cancer immunotherapy.Such is the case for breast, bladder, ovary, uterine, colon, lung,brain, prostate and head and neck tumors. EGF-R expression has proven tobe an indication of bad prognosis in breast cancer (Pérez R et al.(1984) Breast Cancer and Treatment 4:189-193). While the role of EGFRand its ligands in tumor growth is not yet known, there are suggestionsthat EGF-R expression in tumor cells induces a mechanism for autocrinestimulation that leads to uncontrolled proliferation of those cells(Schlessinger J et al. (1983) Crit Rev Biochem 14 (2):93-111).

[0004] The main ligands of this system are the Epidermal Growth Factor(EGF) and the Transforming Growth Factor alpha type (TGFalpha). Thereare other ligands belonging to the EGF superfamily, like: amphireguline(AR), cryto-1 (CR1), Heparin Growth Factor, betacellulin, epiregulin,and others. EGF is a 53 amino acid polypeptide with a molecular weightof 6045 Da, which is mitogenic for cells of epithelial origin. Itsaction is mainly paracrine through its binding to EGF-R.

[0005] TGF alpha is a 50 amino acid polypeptide able to compete with EGFfor binding to EGF-R. Anti-EGF antibodies are not able to recognize TGFalpha (Todaro G J et al. (1976), Nature 264:26-31), meaning that bothgrowth factors are two immunologically different entities.

[0006] The EGFR—ligand system has been the target of passiveimmunotherapy (PI) using monoclonal antibodies(Mab) against EGF-R, innative form, associated with drugs, toxins, or radioactive isotopes(Vollmar A M et al. (1987) J Cell Physiol 131:418-425) in tumors withhigh expression of this receptor. These antibodies have been selected bytheir capacity to inhibit the binding of EGF to it receptor(neutralizing antibodies). Several clinical trials with Mabs are beingcarried out and some have shown promising results as it is the case ofPhase II clinical trials with the Mab C225 in breast, pancreatic andrenal cancer, in addition to Phase III trials in head and neck cancer(Mendelsohn, J et al. (1999) American Society of Clinical OncologyMeeting). Other Phase II clinical trials showing good results have beencarried out with the Mab IOR-R3 in lung tumors (Crombet T et al. (2000)Cancer Biotherapy and Biopharmaceutical, manuscript accepted forpublication).

[0007] Passive immunotherapy with the IOR-R3 Mab (EP586002B1), specificagainst the EGF-R, has demonstrated that the specific binding of theIOR-R3 to the receptor inhibits EGF/EGF-R binding, with subsequentinhibition of EGFR autophosphorylation. In turn, passive immunotherapywith IOR-R3 inhibited the growth of human tumor cells in nude mice, andit has reduced the rate of tumor growth in some patients in clinicaltrials. This system has also been target of specific active cancerimmunotherapy. One example is the use of a vaccine composed one of themain ligands of EGF-R, EGF, coupled to a carrier protein (U.S. Pat. No.5,894,018). This vaccine is able to induce a specific antibody responseagainst autologus EGF, to inhibit EGF/EGF-R binding, thus blockingproliferation mechanisms induced by this binding. Pre-clinical studieshave shown that mice immunized with autologus EGF coupled to a carrierprotein and administered with a useful adjuvant, increases survival ofmice transplanted with Ehrlich Ascitic Tumor (EAT) cells (González G etal. (1996) Vaccine Research 5(4):233-243; Gonzáalez G et al (1997)Vaccine Research 6(2):9 1-100).

[0008] Results from a Phase I clinical trial have been reported for avaccine containing human recombinant EGF, demonstrating theimmunogenicity and safety of vaccination (Gonzáalez G et al (1998),Annals of Oncology 9:1-5).

[0009] Another example of active specific immunotherapy of cancer inthis system is a vaccine composition containing EGF-R, proteoliposomesderived from an external membrane protein complex of Neisseriameningitidis and a ganglioside that associate specifically with thisreceptor forming membrane molecular complexes (Patent deposited in Cuba,priority date Jun. 12, 2000).

[0010] Likewise, vaccines containing other EGF-R ligands, such as TGFalpha alone or combined with EGF and coupled to a carrier protein, havebeen developed ( Patents Requested in Cuba, priority date Jun. 12,2000).

[0011] In the present invention the use of combined immunotherapies isproposed, directed either against receptors with tyrosine kinaseactivity (RTK) or against their ligands,. This combination has theobject of potentiating the observed effect when applying, in anindependent way, different forms of immunotherapy described in the stateof the art, directed alone against some of the receptor/ligand systems.This potentiation is justified for the combined blockade of both,ligands and receptor, in a treatment method that includes bothprinciples.

DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention is related to immunotherapeuticcombinations and treatment methods to inhibit growth of tumor cells toeliminate those cells, based on the blockade of RTK receptors and itsligands. This blockade can be achieved, among other approaches, usingcombination, simultaneous or sequential, of active immunotherapies(therapeutic vaccines) and passive immunotherapies (Mab) directed togrowth factors (i.e. EGF, TGFa) and its receptors (i.e. EGF-R).

[0013] The blockade of growth factors or of their receptors causesinhibition of cellular proliferation. In this invention we show thatsimultaneous blockade of ligands and/or receptors potentiate theinhibition effect on cellular proliferation. This therapeutic concept isof great importance for treatment of malignant tumors, which arefundamentally caused by an increase in the rate of cellularproliferation.

[0014] Immunotherapeutic combinations are described that cause theblockade of RTK receptors and/or their ligands, by means of active andpassive immunotherapeutical combinations. The referred procedures can beapplied to patients with tumors of epithelial origin that over-expressEGF-R, in different clinical stages.

[0015] The combination of active and passive immunotherapy can besimultaneous or sequential, independent of the therapeutic procedureused in patients with advanced disease, or as adjuvant therapy.

[0016] In cases of advanced disease, the proposed therapeuticcombination is passive immunotherapy with Mab that recognizes the RTKreceptor and/or Mab that recognizes ligands of this receptor, incombination with an onco-specific therapy of choice, as first linetherapy, followed by active immunotherapy using vaccines directedagainst the ligands of the receptor and/or to the receptor, to maintainthe theraputic effect. In cases of adjuvant therapy the proposedtherapeutical combinations are:

[0017] 1. Passive immunotherapy with Mab that recognize either, the RTKreceptor and/or its ligands with active immunotherapy using vaccinesdirected to the receptor's ligands or to the receptor itself.

[0018] 2. Passive immunotherapy with Mab that recognize either, the RTKreceptor or its ligands as attack therapy, followed by activeimmunotherapy with vaccines directed to the receptor's ligands or to thereceptor itself, as maintenance treatment.

[0019] PROCEDURE 1: Therapeutic combination including passiveimmunotherapy with Mab that recognize the RTK receptor (i.e.EGF-R)and/or the receptor's ligands (i.e.EGF, TGF alpha), followed by activetherapy with vaccines directed to the receptor and/or its ligands, to beapplied in patients with advanced stage epithelial tumors.

[0020] This will be administered to patients with advanced cancer whoare not eligible for any other onco-specific therapy.

[0021] The first treatment step will be passive immunotherapy with Mabthat recognizes the RTK receptor (i.e.EGF-R), with the property ofinhibiting this receptor and/or Mab that recognize the receptors ligands(i.e. EGF, TGF alpha). This will be an acute therapy aimed at the goalof tumor remission, and can be used together with the establishedonco-specific treatment for this stage of disease.

[0022] This will be followed by active immunotherapy using vaccines thatinduce receptor blocking antibodies (i.e.anti-EGF-R) and/or ligandblocking antibodies (i.e.anti-EGF anti-TGF alpha), with the objective ofmaintaining disease stabilization for longer periods, to avoid newmetastates.

[0023] The procedure consists of administration to patients in advancedstages of tumors of epithelial origin, of between 4 and 20 doses ,ranging between 100 and 400 mg of a Mab that recognizes and inhibitsEGF-R, and/or MAb that recognizes the receptor's ligands. The timebetween doses will be between 6 to 10 days. The complete treatment canlast between 1 to 24 months, concomitant with the establishedonco-especific therapy. The treatment will continue up to partial orcomplete tumor regression or up to the point where an adverse reactionoccurs that requires treatment cessation.

[0024] Between 1 and 4 weeks after this treatment, immunizationschedules will be initiated with vaccines directed against EGF-R or itsligands (i.e.EGF , TGFalpha) coupled to a carrier protein (i.e.P64KNeisseria meningitides recombinant protein) and administered in anadequate adjuvant i.e alum (between 1 and 2 mg/dose) or Montanide ISA 51(between 0.6 and 1.2 ml/dose). Each dose contains between 50 and 800 ugof active ingredient (receptor or ligand) coupled to the carrierprotein, in a final volume of between 0.6 and 5 mL. The immunizationschedule is 5 to 8 initial immunizations for response induction, givenevery 7 to 14 days. Immunizations can be preceded by administration ofcyclophosphamide , between 100 and 500 mg/m² of body weight,administered 2 to 4 days before the 1^(rst) immunization. Vaccines canbe formulated in any other vaccine vehicle (i.e.liposomes, DNA vaccines,viral vectors).

[0025] Vaccines can be formulated as independent products or as a uniquevaccine formulation. In this period, blood will be extracted frompatients in order to measure biochemical blood markers and specificantibody titers against the ligand or receptor to which the vaccine isdirected. Extractions will be done weekly or monthly.

[0026] Subsequently, re-immunizations will be done if antibody titersdecrease, every 1 to 4 months for a period of 1 to 2 years.

[0027] PROCEDURE 2: Immunotherapeutic combination including passiveimmunotherapy with Mab that recognizes a RTK receptor (i.e.EGF-R) and/orit ligands (i.e. EGF, TGF alpha) together with active immunotherapy withvaccines directed against the receptor and/or its ligands, as adjuvanttreatment.

[0028] Passive treatment with Mab recognizing a RTK receptor (i.e.EGF-R)inhibiting its activity and/or Mab recognizing receptor's ligands(i.e.EGF, TGF alpha), together with an active treatment with vaccinesthat induces an antibody response that blocks the receptor and/or itsligands, will be administered to patients immediately after diagnosisand/or surgical treatment.

[0029] Those treatments, administered together, will have a synergisticeffect, enabling a higher percentage of regression and/or clinicaldisease stabilization.

[0030] Patients with tumors of epithelial origin are amenable to thistreatment, that consists of between 4 to 20 doses, ranging between 100and 400 mg, of Mab recognizing and inhibiting RTK receptors and/or itligands. The time between doses will be between 6 to 10 days and thetreatment can last between 1 to 24 months. The treatment will continueuntil partial or complete tumor regression or up to the point where anadverse reaction occurs that requires treatment cessation.

[0031] Concomitant immunizations will be administered with vaccinesaccording to the schedule described in procedure #1.

[0032] PROCEDURE 3:

[0033] Immunotherapeutic combination including passive immunotherapywith Mab recognizing RTK receptors (i.e.EGF-R) and/or its ligands (i.e.EGF, TGF alpha), followed by active immunotherapy with vaccines directedagainst the receptor and/or it ligands, to be applied as adjuvanttherapy.

[0034] This will be applied to patients immediately after diagnosisand/or surgical treatment. The goal of this treatment is to use acutetherapy to obtain tumor remission, via initial passive immunotherarywith Mab recognizing and inhibiting RTK receptors (i.e.EGF-R) and/or Mabrecognizing its ligands (i.e. EGF, TGF alpha).Subsequently, activeimmunotherapy will be initiated using vaccines inducing blockingantibodies against the receptor (i.e.EGF-R) or it ligands (i.e.EGF, TGFalpha). The aim of the 2^(nd) treatment is to obtain a longer period offreedom from disease, to avoid the appearance of new metastates.

[0035] The procedure consists of administration to patients at advancedstages of cancer of epithelial origin, from 4 to 20 doses of between 100and 400 mg of Mab that recognizes and inhibits the EGF-R and/or itsligands. The time between doses will be between 6 to 10 days and thetreatment duration can be between 1 to 24 months. The treatment willcontinue until partial or complete tumor regression, or until anyadverse reaction occurs that requires treatment cessation.

[0036] Between 1 to 4 weeks after the end of treatment, immunizationschedules will begin with vaccines directed against the EGF-R or someEGF-R ligand (i.e. EGF, TGF alpha), according to the schedule describedin procedure #1.

EXAMPLES Example 1: Immunization Schedule with EGF Vaccine in CancerPatients, Using Alum as Adjuvant

[0037] With the main goal of demonstrating immunogenicity and safety ofEGF, a clinical trial was performed in which 10 patients were immunizedwith an EGF Vaccine (U.S. Pat. No. 5,894,018), using P64K as carrierprotein and alum as adjuvant,.

[0038] Patient 1.1 (MMG) was included in the trial with a diagnosis ofmetastasic epidermoid carcinoma of the lung, with progressive disease,and not eligible for any other onco-specific treatment.

[0039] The patient was immunized following a schedule of 5 initial doseof the vaccine, containing 50ug of EGF and 2 mg alum, administered ondays 1, 7, 14, 21 and 51.

[0040] Blood extraction was performed on days 0, 15, 30, 45, 60 andmonthly thereafter for blood biochemical measurements and forEGF-specific antibodies. Antibody titers were measured by means of anELISA test, antibody titers being determined as the maximal seradilution that gives a positive result in the ELISA test. (O.D valuesequal or higher 2 times the blank).

[0041] Re-immunization was performed using the same vaccine dose when adecrease in antibody titers was detected.

[0042] Patient MMG developed an anti-EGF antibody response with maximumtiters up to 1:8000. The kinetics of the antibody response is shown inFIG. 1.

[0043] After the beginning of the vaccination schedule the patientshowed clinical and radiological stabilization of disease for 15 months.The patient died 23.2 months after the first vaccination.

Example 2 Immunization Schedule with EGF Vaccine in Cancer Patients,Using Montanide ISA 51 as Adjuvant

[0044] With the main goal of demonstrating immunogenicity and safety ofEGF using P64K as a carrier protein and Montanide ISA 51 as an adjuvant,a clinical trial was performed in which 10 patients were immunized.

[0045] Patient 2.1 (AMG) was included in the trial with a diagnosis ofepidermoid carcinoma of the lung, with progressive disease, beingineligible for any other onco-specific treatment. The patient wasimmunized according to a schedule of 5 initial doses of the vaccinecontaining 50 ug of EGF in 0.6 mL total volume, emulsified with 0.6 mLof Montanide ISA 51 immediately before use, and administered on days 1,7, 14, 21 and 51.

[0046] Blood extractions were performed on days 0, 15, 30, 45, 60 andmonthly thereafter for blood biochemical measurements and measurement ofspecific anti-EGF antibodies.

[0047] The antibody titers were measured by means of an ELISA test,antibody titers being determined as the maximal sera dilution that givesa positive result in the ELISA test. (O.D values equal or higher 2 timesthe blank).

[0048] Re-immunization was performed using the same vaccine dose when adecrease in antibody titers was detected.

[0049] Patient AMG developed an anti-EGF antibody response with maximumtiters of up to 1: 32000, with a kinetics of response shown in FIG. 2.

[0050] After the beginning of the vaccination schedule, the patientshowed stabilization of disease for 12 months, at which point clinicaland radiological tumor regression was diagnosed.

[0051] On the 14^(th) month after the beginning of vaccination, a 2^(nd)primary tumor appeared. The patient died 18 months after inclusion froma surgical complication of this 2^(nd) tumor.

Example 3 Immunization Schedule in Cancer Patients, with EGF Vaccine,Using Alum as Adjuvant and Low Dose Cyclophosphamide Pre-treatment

[0052] A clinical trial was carried out in which 10 patients wereimmunized with the main goal of demonstrating immunogenicity and safetyof the EGF Vaccine using P64K as carrier protein and alum as adjuvantafter cyclophosphamide pre-treatment.

[0053] Patient 3.1, FNR, was included in the trial with a diagnosis ofepidermoid carcinoma of the lung, with progressive disease, beingineligible for any other onco-specific treatment. The patient wastreated with cyclophosphamide (100 mg/m² of body surface), 3 days beforethe first immunization of the EGF Vaccine. The vaccination schedule was5 doses of the vaccine composition, containing 50 ug of EGF and 2 mg ofalum, administered on days 1, 7, 14, 21 and 51.

[0054] Blood extractions were performed on days 0, 15, 30, 45, 60 andthen monthly for blood chemistry and specific anti-EGF antibodydeterminations.

[0055] Antibody titers were measured by means of an ELISA test, antibodytiters being determined as the maximal sera dilution that gives apositive result in the ELISA test. (O.D values equal or higher 2 timesthe blank).

[0056] Re-immunization was performed using the same vaccine dose, when adecrease in antibody titers were detected.

[0057] The patient developed an anti-EGF antibody response with maximumtiters up to 1:8000, as shown in FIG. 4.

[0058] After the beginning of the vaccination schedule, the patientshowed disease stabilization for 19 months.

Example 4 Immunization Schedule with EGF Vaccine in Cancer Patients,Using Montanide ISA 51 as Adjuvant and Cyclophosphamide Pre-treatment

[0059] A clinical trial was carried out in which 10 patients wereimmunized with the main goal of demonstrating immunogenicity and safetyof the EGF Vaccine, using P64K as carrier protein and and Montanide ISA51 as adjuvant after cyclophosphamide pre-treatment. Patient 4.1, JPG,was included in the trial with a diagnosis of non small cell lungadenocarcinoma, with progressive disease, being ineligible for any otheronco-specific treatment.

[0060] The patient was treated with cyclophosphamide (100 mg/m² of bodysurface), 3 days before the first immunization of the EGF Vaccine. Thevaccination schedule was 5 doses of the vaccine composition, containing50 ug of EGF in 0.6 mL total volume, emulsified with 0.6 mL of MontanideISA 51 immediately before use, administered on days 1, 7, 14, 21 and 51.

[0061] Blood extractions were performed on days 0, 15, 30, 45, 60 andthen monthly for blood chemistry and specific anti-EGF antibodydeterminations. Antibody titers were measured by means of an ELISA test,antibody titers being determined as the maximal sera dilution that givesa positive result in the ELISA test. (O.D values equal or higher 2 timesthe blank).

[0062] Re-immunization was performed , using the same vaccine dose, whena decrease in antibody titers was detected.

[0063] Patient JPG developed an anti-EGF antibody response with maximumtiters up to 1:400000, as shown in FIG. 5.

[0064] After the beginning of the vaccination schedule the patientshowed disease stabilization for 6 months.

Example 5 Immunogenicity of EGF Vaccination and its Relationship toDisease Stabilization in Patients with Cancer

[0065] A Phase I trial in 20 patients was performed in which patientswere randomized to one of two groups using different adjuvants..

[0066] Ten patients at stages III or IV of Non Small Cell Lung cancer(NSCLC), were treated with 5 initial doses of vaccine compositioncontaining 50ug of EGF and 2 mg of alum, administered on days 1, 7, 14,21 and 51.

[0067] The other 10 patients (NSCLC, stages III or IV), were immunizedwith 5 doses of the vaccine composition containing 50 ug of EGF, in atotal volume of 0.6 mls, emulsified with the same volume (0.6 mL) ofMontanide ISA 51.

[0068] Antibody titers were measured by means of an ELISA test, withantibody titers determined as the maximal sera dilution that gives apositive result in the ELISA test. (O.D values equal or higher 2 timesthe blank).

[0069] In this trial, 50% of patients developed an anti-EGF antibodyresponse with antibody titers of 1:4000 or higher (, Good AntibodyResponders, GAR group) and 50% antibody titers below 1:4000 (BadAntibody Responders, BAR group).

[0070] In the GAR group, 87.5% of patients showed clinical andradiological disease stabilization for at least3 months after thebeginning of treatment.

[0071] In the BAR group, only 11,1% of patients showed thisstabilization profile (Table 1).

[0072] These data demonstrate the relationship between anti-EGF antibodylevels and tumor stabilization. CHART 1 Relationship of antibodyresponses and clinical and radiological disease stabilization. Diseasestabilization for at least 3 months after % of patients beginningtreatment. GAR 50% 87.5% BAR 50% 11.1%

Example 6 Immunogenicity of EGF Vaccination and Relationship to Survivalof Cancer Patient Subjected to This Treatment

[0073] Forty stage III /IV NSCLC patients were treated, in groups of 10,with the schedules detailed in examples 1,2,3 and 4.

[0074] They were characterized as GAR and BAR according to criteriaexposed in example 6. Of the total of patients treated with thepreviously described schedules, 50% turned out to be GAR and 50% BAR.

[0075] When survival patterns were compared between GAR and BARpatients, a statistically significant difference was observed, with amean survival of 9.1 months for GAR and a mean survival of 4.5 monthsfor BAR (p <0.02). This result is showed in FIG. 6.

Example 7 Therapeutic Effect of the Combination of Radiotherapy and MabIOR-R3

[0076] Patient RML, diagnosed with stage IV language base epidermoidcarcinoma, was included in the clinical trial using the combination ofradiotherapy (RTP) and IOR-R3.

[0077] The patient received 200 mg of Mab once a week for 6 weeks. Theaccumulated dose of Mab was 1200mg and the total radiation dose was 60Gy.

[0078] When the combination therapy was complete the patient showedcomplete remission of the primary tumor and its metastases (FIG. 7).This response was maintained for more than 13 months.

Example 8 Therapeutic Effect of the Combination of Radiotherapy and MabIOR-R3

[0079] Patient EPG, diagnosed with stage III tonsil epidermoid carcinomawith cervical adenopathies, was included in the clinical trial using thecombination of radiotherapy (RTP) and IOR-R3. The patient received 200mgof Mab once a week for 6 weeks and a total radiation dose of 64 Gy.

[0080] After treatment, this patient showed complete remission of thetumor lesion (FIG. 8).

[0081] The response was maintained for more than 13 months.

Example 9 Therapeutic Effect of the Combination of Radiotherapy and MabIOR-R3

[0082] Patient CHA, diagnosed with a stage IV tonsil tumor, withbilateral cervical adenopathies, was included in the clinical trialusing a combination of radiotherapy (RTP) and IOR-R3. The patientreceived 400 mg of Mab once a week for 6 weeks, for an accumulated doseof 2400 mg. Concomitantly, the patient received a total radiation doseof 64 Gy.

[0083] When concluding the treatment this patient was in completeremission of the primary tumor and the loco-regional metastasis (FIG.9). The response was maintained for 12 months.

Example 10 Evaluation, in Nude Mice, of Passive Therapy Using aCombination of Anti-EGF-R Antibody (IOR-R3) and an Anti-EGF-R LigandMonoclonal (EGF-1)

[0084] Evaluation of the anti-tumor effect in relation to theadministered doses.

[0085] This experiment also simulates the possible effect of combinedadministration of the anti-EGF-R Mab and an EGF vaccine. The vaccinecauses an anti-EGF antibody response with the same effect of passiveadministration of Mab with that specificity, with the additionaladvantage that, the achieved antibody response can be maintained overtime, as shown in examples 1, 2, 3 and 4 (kinetics of anti-EGF antibodytiters in immunized patients)

[0086] Seven different groups of athymic mice, with NMRI genetic origin(outbred population), were immunized with:

[0087] Group 1:10 doses of 0.5 mg of the EGF-1 Mab, intraperitonealroute, daily frequency.

[0088] Group 2: 10 doses of 1 mg of the EGF-1 Mab, intraperitonealroute, daily frequency.

[0089] Group 3: 10 doses of 0.5 mg of the IOR-R3 Mab, intraperitonealroute, daily frequency.

[0090] Group 4: 10 doses of 1 mg of the IOR-R3 Mab, intraperitonealroute, daily frequency.

[0091] Group 5: 10 doses of Phosphate Buffered Saline (PBS),intraperitoneal route, daily frequency (negative control).

[0092] Group 6: 10 doses of 0.5 m g EGF-1 Mab combined with 0.5 mgIOR-R3 Mab, intraperitoneal route, daily frequency.

[0093] Group 7: 10 doses of 1 m g EGF-1 Mab combined with 1 mg IOR-R3Mab, intraperitoneal route, daily frequency.

[0094] On the day of initiation of treatment with Mabs mice weretransplanted with 1 ×10⁶ H125 human tumor cells. This cell lineover-expresses the EGF-R.

[0095] Results are shown in FIGS. 10 and 11. The anti-tumor effect waspotentiated when both treatments were combined, increasing withincreased doses.

Example 11 Schedule of Combined Mab IOR-R3/EGF Vaccine Treatments inPatients With Advanced Stage Tumors

[0096] Patient ARP, diagnosed with epidermoid carcinoma of the head andneck, received sequential treatment of Mab IOR-R3 and EGF Vaccine.

[0097] The patient received 200 mg of Mab once a week for 6 weeks, incombination with a total of 30 doses of radiotherapy , 5 doses per weekfor 6 weeks, for an accumulated radiation dose of 60 Gy.

[0098] When concluding the treatment, the patient was in completeremission of the primary tumor.

[0099] An immunization schedule with the EGF Vaccine began one monthafter conclusion of the treatment with the Mab. The patient received 5doses of 50 ug of EGF conjugated to protein P64k, in a total volume of0.6 mls, emulsified with 0.6 ml of Montanide ISA 51 immediately beforeuse. The immunizations were carried out on days 1,7,14,21 and 51.

[0100] The patient remains in the follow-up period.

Example 12 Schedule of Combined Mab IOR-R3/EGF Vaccine Treatments inPatients With Advanced Stages Tumors

[0101] Patient MRM, diagnosed with epidermoid carcinoma of the lung, wassubjected to surgical intervention. One month after the surgery thepatient began a combined treatment of passive immunotherapy with MabIOR-R3 concomitantly with the EGF vaccine.

[0102]FIG. 12 details the schedule of dose intervals. The patient is infollow up.

BRIEF DESCRIPTION OF FIGURES

[0103]FIG. 1: Kinetics of anti-EGF antibody response in patient MMG,immunized as detailed in example 1. Arrows indicate times ofre-immunizations.

[0104]FIG. 2: Kinetics of anti-EGF antibody response in patient AMC,immunized as detailed in example 2. Arrows indicate times ofre-immunizations.

[0105]FIG. 3: Tumor regression observed in patient AMC. The tumor massis seen on the left at the start of treatment. On the right of thefigure it can be seen that 12 months after the start of treatment thetumor mass disappeared.

[0106]FIG. 4: Kinetics of anti-EGF antibody response in patient FNR,immunized as detailed in example 3. Arrows indicate times ofre-immunizations.

[0107]FIG. 5: Kinetics of anti-EGF antibody response in patient JPG,immunized as detailed in example 4. Arrows indicate times ofre-immunizations.

[0108]FIG. 6: Kaplan-Maier survival curves of groups of patients withhigh anti-EGF antibody response (GAR) and with low anti-EGF antibodyresponses (BAR), as well as that of a historical control group. As canbeseen, GAR is associated with a significant increase in survivalcompared with either BAR or with historical controls.

[0109]FIG. 7: Graphic demonstration of tumor remission in patient RML,treated as detailed in example 7.

[0110]FIG. 8: Graphic demonstration of tumor regression in patient EPG,treated as detailed in example 8.

[0111]FIG. 9: Graphic demonstration of the tumor in patient CHA, treatedas detailed in example 9.

[0112]FIG. 10: Groups of mice immunized with 0.5 mg of both Mabs IOR-R3and EGF-1, and with the combination of 0.5 mg of IOR-R3+0.5 mg of EGF1,as detailed in example 10. A synergistic effect on decreased tumorgrowth was observed in the group treated with the combination of bothMabs.

[0113]FIG. 11: Groups of mice immunized with 1 mg of both Mabs IOR-R3and EGF-1, and with the combination of 1 mg of IOR-R3+1 mg of EGF1, asdetailed in example 10. A synergistic effect on decreased tumor growthwas observed in the group treated with the combination of both Mabs.

[0114]FIG. 12: Combined treatment of Mab IOR-R3 and the EGF Vaccine:Arrows above the time axis indicate the day of Mab administration (days1,7,14,21,28 and 35) and arrows below the time axis indicate the day ofimmunization with the EGF Vaccine (days 2,8,15,22, and 52).

1. A combination useful for immunotherapy, where this combination has aneffect on growth and/or proliferation of cells, whose growth isdependent on the interaction between a receptor and its ligand, in thereceptor tyrosine kinase system (RTK), this combination includes: a. —Anantibody against a RTK receptor. b. —A vaccine in which the activeprinciple is the RTK receptor, and that induces antibodies against thisreceptor.
 2. Immunotherapy combination according to claim 1 where theRTK is the EGF receptor.
 3. Immunotherapy combination according to claim2 where the vaccine is directed against the EGF receptor. 4.Immunotherapy combination according to claim 2 where the antibodyagainst the RTK receptor is an antibody against the EGF receptor. 5.Immunotherapy combination according to claim 4 where the antibodyagainst the RTK receptor is a humanized antibody against the EGFreceptor.
 6. Immunotherapy combination according to claim 5 where thehumanized antibody against the EGF receptor is IOR R3.
 7. A treatmentcombination useful for immunotherapy, where this combination has aneffect on growth and/or proliferation of cells, whose growth isdependent on the interaction between a receptor and its ligand, in thesystem of receptor tyrosine kinases (RTK), this combination including:a. —An antibody against the ligands of the RTK receptor and b. —Avaccine whose active principle is (are) the RTK receptor's ligand(s) andthat induces antibodies against this (those) ligand(s).
 8. A therapeuticcombination according to claim 7 where the RTK receptor's ligand is EGF.9. A therapeutic combination according to claim 8 where the vaccine iscomposed of conjugated proteins P64K and EGF.
 10. A therapeuticcombination according to claim 7 where the RTK receptor ligand isTGF-alpha.
 11. A therapeutic combination according to claim 10 where thevaccine is composed of conjugated proteins P64K and TGF alpha.
 12. Acombination useful for immunotherapy, where this combination has aneffect on growth and/or proliferation of cells, whose growth isdependent on the interaction between a receptor and its ligand, in thesystem of receptor protein tyrosine kinases (RTK), this combinationincludes: a. —A first agent selected from one of the antibodies againstthe RTK receptor and of a vaccine where the active principle is the RTKreceptor that induces antibodies against this receptor, and b. —A secondagent selected from of one of the antibodies against the ligands of theRTK receptor and of a vaccine where the active principle is this ligand,which induces antibodies against said ligand.
 13. An immunotherapycombination according to claim 12, where the first agent is an antibodyagainst the RTK receptor.
 14. An immunotherapy combination according toclaim 13 where the antibody against the RTK receptor is an antibodyagainst the EGF receptor.
 15. An immunotherapy combination according toclaim 14 where the antibody against the EGF receptor is a monoclonalantibody.
 16. An immunotherapy combination according to claim 15 wherethe antibody against the EGF receptor is a humanized antibody.
 17. Animmunotherapy combination according to claim 16 where the antibodyagainst the EGF receptor is IOR R3.
 18. An immunotherapy combinationaccording to claim 12, where the first agent is a vaccine whose activeprinciple is an RTK receptor.
 19. An immunotherapy combination accordingto claim 18, where the first agent is a vaccine whose active principleis the EGF receptor.
 20. An immunotherapy combination according to claim12 where the second agent is an antibody against an RTK receptor ligand.21. An immunotherapy combination according to claim 20 where theantibody against the RTK receptor's ligand is an antibody against EGF.22. An immunotherapy combination according to claim 20 where theantibody against the RTK receptor is an antibody against TGF-alpha. 23.An immunotherapy combination according to claim 12, where the secondagent is a vaccine whose active principle is an RTK receptor's ligand.24. An immunotherapy combination according to claim 23 where the vaccinecontains EGF as active principle.
 25. An immunotherapy combinationaccording claim to 24 where the vaccine contains conjugated proteinsp64K and EGF as active principle.
 26. An immunotherapy combinationaccording to claim 23 where the vaccine contains TGF-alpha as activeprinciple.
 27. An immunotherapy combination according to claims 1 to 26inclusive, whose combination consists of a mixture of reagentscontaining independent doses of effective formulations, either of Mab orvaccines, where the combination of those independent formulationsinduces decreased growth of tumors that over-express EGF-R.
 28. Animmunotherapy combination according to claim 27, whose combinationconsists of a mixture of reagents containing independent doses ofeffective formulations, either of Mab against the EGF receptor and itsligands or of vaccines with EGF-R and its ligands (EGF, TGF alpha) asactive principle, and where the combination of those independentformulations induces decreased growth of tumors that over-express EGF-R.29. A method to control growth and/or proliferation of cells whosegrowth is dependent on the interaction between a receptor and itsligand, in the receptor tyrosine kinase (RTK) system, this methodincluding the treatment with one of the therapeutic combinations definedin any one of the preceding claims.
 30. A method according to claim 29that includes the simultaneous treatment with agents against RTKreceptors and their ligands.
 31. A method according to claim 29 thatincludes the simultaneous treatment with vaccines and antibodies.
 32. Amethod according to claim 29 that includes the treatment at the firststage with this antibody and at a second stage with this vaccine.
 33. Amethod according to claim 29 that includes the treatment at a firststage with this vaccine and at a second stage with this antibody.